RESUMO
Prebiotics can modulate the gut microbial community composition and function for improved (gut) health and increase resilience against infections. In vitro models of the gut facilitate the study of intervention effects on the gut microbial community relevant to health. The mucosa-associated gut microbiota, which thrives in close contact with the host plays a pivotal role in colonization resistance and health. Therefore, we here introduce the Mi-screen, an experimental approach implementing a 96-well plate equipped with a mucus agar layer for the additional culturing of mucosa-associated microbiota in vitro. In this study, we screened the effects of 2'-Fucosyllactose (2'-FL), fructooligosaccharides (FOS), and inulin within a complex microbiota without and with infection with the C. difficile strains ATCC 43599 (Ribotype 001) or ATCC BAA-1870 (Ribotype 027). We analyzed the microbial community composition and short-chain fatty acid levels after 48 h of incubation. The inclusion of an additional substrate and surface in the form of the mucus agar layer allowed us to culture a microbial richness ranging between 100-160 in Chao index, with Shannon indices of 5-6 across culture conditions, indicative of a microbial diversity of physiological relevance. The mucus agar layer stimulated the growth of characteristic mucosa-associated bacteria such as Roseburia inulinovorans. The prebiotic interventions affected luminal and mucosal microbial communities cultured in vitro and stimulated short-chain fatty acid production. FOS, inulin and 2'-FL promoted the growth of Bifidobacterium adolescentis within the mucosa-associated microbiota cultured in vitro. When spiking the untreated conditions with pathogenic C. difficile, the strains thrived within the luminal and the mucosal sample types, whereas prebiotic treatments exhibited inhibitory effects on C. difficile growth and prevented colonization. In conclusion, the Mi-screen facilitates the screening of luminal and mucosa-associated gut microbial community dynamics in vitro and therefore fills an important gap in the field of in vitro modeling.
RESUMO
Background: The role of the vulvar microbiome in the development of (pre)malignant vulvar disease is scarcely investigated. The aim of this exploratory study was to analyze vulvar microbiome composition in lichen sclerosus (LS) and vulvar high-grade squamous intraepithelial lesions (HSIL) compared to healthy controls. Methods: Women with vulvar lichen sclerosus (n = 10), HSIL (n = 5) and healthy controls (n = 10) were included. Swabs were collected from the vulva, vagina and anal region for microbiome characterization by metagenomic shotgun sequencing. Both lesional and non-lesional sites were examined. Biophysical assessments included trans-epidermal water loss for evaluation of the vulvar skin barrier function and vulvar and vaginal pH measurements. Results: Healthy vulvar skin resembled vaginal, anal and skin-like microbiome composition, including the genera Prevotella, Lactobacillus, Gardnerella, Staphylococcus, Cutibacterium, and Corynebacterium. Significant differences were observed in diversity between vulvar skin of healthy controls and LS patients. Compared to the healthy vulvar skin, vulvar microbiome composition of both LS and vulvar HSIL patients was characterized by significantly higher proportions of, respectively, Papillomaviridae (p = 0.045) and Alphapapillomavirus (p = 0.002). In contrast, the Prevotella genus (p = 0.031) and Bacteroidales orders (p = 0.038) were significantly less abundant in LS, as was the Actinobacteria class (p = 0.040) in vulvar HSIL. While bacteria and viruses were most abundant, fungal and archaeal taxa were scarcely observed. Trans-epidermal water loss was higher in vulvar HSIL compared to healthy vulvar skin (p = 0.043). Conclusion: This study is the first to examine the vulvar microbiome through metagenomic shotgun sequencing in LS and HSIL patients. Diseased vulvar skin presents a distinct signature compared to healthy vulvar skin with respect to bacterial and viral fractions of the microbiome. Key findings include the presence of papillomaviruses in LS as well as in vulvar HSIL, although LS is generally considered an HPV-independent risk factor for vulvar dysplasia. This exploratory study provides clues to the etiology of vulvar premalignancies and may act as a steppingstone for expanding the knowledge on potential drivers of disease progression.